CN106399564B

CN106399564B - Application of ERCC8 gene in detection of congenital cataract combined with keratoconus

Info

Publication number: CN106399564B
Application number: CN201611038913.8A
Authority: CN
Inventors: 郝晓丹; 赵晓雯; 李素霞; 田晓
Original assignee: SHANDONG EYE INSTITUTE
Current assignee: SHANDONG EYE INSTITUTE
Priority date: 2016-11-11
Filing date: 2016-11-11
Publication date: 2020-09-15
Anticipated expiration: 2036-11-11
Also published as: CN106399564A

Abstract

The invention aims to provide application of an ERCC8 gene in preparation of a product for detecting congenital cataract combined with keratoconus, and provides new application of the ERCC8 gene, so that an effective way for performing genetic diagnosis, prenatal gene screening and genetic consultation on congenital cataract combined keratoconus diseases is provided, and application effects show that the SNP locus and detection primers of the gene provided by the invention can be effectively used for clinical patients and fetal villi or amniotic fluid to perform rapid detection on the mutation locus of the congenital cataract combined keratoconus gene.

Description

Application of ERCC8 gene in detection of congenital cataract combined with keratoconus

Technical Field

The invention belongs to the technical field of gene diagnosis products, and particularly relates to application of an excision repair cross complementation group 8(ERCC8) in preparation of a gene diagnosis product for detecting congenital cataract combined with conical cornea.

Background

Congenital cataract (congenital cataract) is a serious blinding lens disease, and is a disease in which the transparency of the lens itself is reduced due to abnormal metabolism of the lens in the embryonic stage. The disease is one of the main causes of blindness of children, has the incidence rate of about 0.01-0.06 percent and accounts for the second place of blindness-causing eye diseases of children. Congenital cataract can occur independently, and can also be used as a concomitant symptom of eye or general syndrome, both eyes are often involved, and anterior segment dysplasia, such as iris defect, Peters abnormity, ectopia pupillae, nystagmus, keratoconus and the like, is often accompanied, and the multiple deformities often cause most patients to lose vision, and is one of important reasons for congenital blindness. Since the vision of infants is developing, and congenital cataract can seriously affect the development of vision, once the best treatment opportunity is missed, the vision is difficult to recover even if an operation is performed, so the life quality of patients can be seriously affected, and the economic and mental burdens are brought to the society and families.

In congenital cataracts, about half of the cases are genetically related and the vast majority of phenotypes are autosomal dominant. To date, thirty or more pathogenic genes and hundreds of mutation sites have been found to be associated with congenital cataract, including crystallin genes, transcription factor genes, connexin genes, transmembrane transporter genes, and the like. The identification of the gene/locus of the congenital cataract has important significance for disclosing the pathogenesis of the congenital cataract and researching the prevention and treatment measures thereof. The congenital cataract has remarkable genetic heterogeneity, and a plurality of candidate pathogenic genes exist, which becomes the bottleneck of the research on the pathogenesis of the congenital cataract and the diagnosis and treatment research on the etiology. The current situation promotes to further discover and understand new pathogenic genes of the disease, and provides a basis for subsequent gene diagnosis, prenatal diagnosis and gene therapy.

Disclosure of Invention

The invention aims to provide application of ERCC8 gene in preparation of a diagnosis product for detecting congenital cataract combined with keratoconus, thereby making up for the defects of the prior art.

The applicant carries out second-generation high-throughput sequencing of 505 genes of monogenic genetic diseases related to abnormal visual system on one patient in a 2-generation autosomal dominant hereditary congenital cataract combined keratoconus family, and finds out the genetic pathogenic mutation of the pathogenic gene ERCC8 of the family through the verification of a first-generation sequencing method, thereby promoting the invention.

The invention firstly provides a new application of ERCC8 gene, which is an application in preparing a diagnosis product for detecting congenital cataract and keratoconus;

the invention also provides a primer pair for detecting the combination of the congenital cataract and the keratoconus, wherein the upstream and downstream primer sequences of the primer pair are SEQ ID NO. 1-24, and the primer information is as follows:

ERCC8-1F:CGGTGTGAGGACACGATA SEQ ID NO:1

ERCC8-1R:AGGGCAGTTATTCTTTGGAG SEQ ID NO:2

ERCC8-2F:AAATACGTTAGGATGTGTGGTA SEQ ID NO:3

ERCC8-2R:CAACATTTGGTAGGTTCGATG SEQ ID NO:4

ERCC8-3F:AGAAATCTCTTTTGCCGTCT SEQ ID NO:5

ERCC8-3R:TATGTGAACCATTCGCTTGA SEQ ID NO:6

ERCC8-4F:GCTGACGTCATAGAGTCTTCA SEQ ID NO:7

ERCC8-4R:ACCACTGTACTGCTTTCACATC SEQ ID NO:8

ERCC8-5F:AAGAAGGGGCAATTCTAGGT SEQ ID NO:9

ERCC8-5R:TGAAGATGTTTGTTGCAGGT SEQ ID NO:10

ERCC6-6F:GCATGGATACAGTGAAAATGTC SEQ ID NO:11

ERCC8-6R:ACACTGATGTGAGTTGCATT SEQ ID NO:12

ERCC8-7F:TTTGGCCTCACTTTCTTCAG SEQ ID NO:13

ERCC8-7R:GACACAACACAGTTCCTCTG SEQ ID NO:14

ERCC8-8F:GGTACAGTCATTGTCCTAGC SEQ ID NO:15

ERCC8-8R:CTTTGTCACATGGATTGCAG SEQ ID NO:16

ERCC8-9F:CATGGCACAATTTCCTTGCT SEQ ID NO:17

ERCC8-9R:GTAGGTAGTGGGTAAGGGTG SEQ ID NO:18

ERCC8-10F:TAGCCAAATTACACTGCCAC SEQ ID NO:19

ERCC8-10R:ATATAACTGGTCTGGCAAGC SEQ ID NO:20

ERCC8-11F:GACATTGACATAGGCTGTGC SEQ ID NO:21

ERCC8-11R:TAGAAGTCACTGTACCATTTGTG SEQ ID NO:22

ERCC8-12F:AGTCTTAGCCTATGGATATCAG SEQ ID NO:23

ERCC8-12R:GTCACAACTGAGGTACAACG SEQ ID NO:24

the invention also provides a kit for detecting the combination of the congenital cataract and the keratoconus, which comprises any one or more of the primer pairs.

The invention also provides an SNP locus related to the congenital cataract combined keratoconus disease, which is deletion mutation from 394 th to 398 th from an initiation codon in an ERCC8 gene coding region, wherein the basic group is TTACA or-;

wherein the primer information for detecting the SNP sites is as follows:

ERCC8-4F:GCTGACGTCATAGAGTCTTCASEQ ID NO:7

ERCC8-4R:ACCACTGTACTGCTTTCACATC SEQ ID NO:8

the invention provides a new application of ERCC8 gene, thereby providing an effective way for performing gene diagnosis, prenatal gene screening and genetic counseling of congenital cataract combined with keratoconus disease, and the application effect shows that the SNP locus and the detection primer of the gene provided by the invention can be effectively used for rapid detection of ERCC8 gene mutation locus of clinical patients and fetal villi or amniotic fluid.

Drawings

FIG. 1: the ERCC8 sequencing map of the patient in the family of congenital cataract and keratoconus of example 1, wherein A: the patient's husband, normal phenotype; b: a patient; c: patient son, disease phenotype. In the family, the coding region of ERCC8 gene of two patients has heterozygous deletion mutation from 394 th to 398 th bases of initiation codon, and the bases are changed from TTACA to-.

Detailed Description

The applicant finds the mutation site of ERCC8 gene in one congenital cataract and keratoconus family and proves that the mutation of the gene is the pathogenic gene of the disease, thereby leading to the invention.

The ERCC8 gene is located on chromosome 5q12.1, can be transcribed into mRNA (NCBI accession No. NM-000082.3) of about 2044bp, and is directly translated into protein consisting of 396 amino acids.

The present invention will be described in detail with reference to examples.

Example 1: screening out mutation sites of ERCC8 gene from congenital cataract combined cone family

1. Extracting peripheral blood genome DNA:

on the basis of meeting the national relevant policy regulations and agreeing with the sampling object, extracting 2-5ml of peripheral venous blood of family members, and putting the peripheral venous blood into an EDTA anticoagulant tube to be frozen at-80 ℃ for later use; after the frozen EDTA anticoagulation blood is melted at room temperature, 500 microliter of the EDTA anticoagulation blood is put into a centrifuge tube, equal volume of TE (pH8.0) is added into the centrifuge tube, the mixture is mixed evenly, the mixture is centrifuged for 10 minutes at 10000rpm at 4 ℃, and the supernatant is discarded.

Mu.l TE, 20. mu.l SDS (10%) and 8. mu.l proteinase K (10mg/ml) were added thereto, mixed well, and placed in a 56 ℃ water bath overnight. The sample was removed from the water bath and the sample was pelleted by instantaneous centrifugation. An equal volume of Tris-saturated phenol (about 300. mu.l) was added to the reaction tube, mixed well, centrifuged at 10000rpm for 10 minutes at room temperature, and the supernatant (about 300. mu.l) was pipetted into a new centrifuge tube. Phenol extraction was repeated once and the supernatant was aspirated into a new centrifuge tube.

Adding equal volume of Tris saturated phenol and chloroform mixed solution (150. mu.l of phenol and chloroform respectively), mixing, centrifuging at room temperature of 10000rpm for 10 minutes, and transferring the supernatant to a new centrifuge tube.

Adding equal volume of Tris saturated phenol, chloroform and isoamyl alcohol mixed solution (100 mul of each of phenol, chloroform and isoamyl alcohol), mixing, centrifuging at room temperature of 10000rpm for 10 minutes, and transferring the supernatant to a new centrifuge tube.

1/10 volumes of 3mol/L sodium acetate (about 30. mu.l) pH5.2 were added, 2 volumes were pre-cooled in 100% ethanol and gently mixed to give a white flocculent precipitate. The DNA was precipitated at the bottom of the tube by centrifugation at 10000rpm for 10 minutes at room temperature, and the supernatant was discarded.

To the DNA precipitation with 70% ethanol, rinsing, room temperature 7000rpm centrifugation for 5 minutes, abandoning the supernatant, placed in room temperature to volatilize the ethanol, finally adding 50 u l TE (pH8.0), 4 degrees overnight dissolved DNA.

And (3) performing agarose gel electrophoresis on the extracted DNA, and performing color comparison at 260nm and 280nm by using an ultraviolet spectrophotometer to detect the purity and the concentration of the DNA.

2. Target sequence capture high-throughput sequencing: the target sequence capturing high-throughput sequencing technology is a novel genome analysis technology, a set of nucleotide probes is used for capturing target sequences on a genome, then universal primers are used for amplifying the captured sequences, and high-throughput bioinformatics analysis is carried out on amplification products. The genome DNA of 1 patient in the family is taken, the probe is applied by Beijing Deyi east transformation medical research center, Inc. to capture the exon regions of 505 genes of the monogenic genetic diseases related to the abnormality of the human visual system, and then the high-throughput sequencing is carried out on the enriched exon libraries. Mutations were filtered through 4 normal human databases: single nucleotide polymorphism database (ftp:// ftp. ncbi. nih. gov/snp/database /), thousand human genome project (ftp:// ftp-trace. ncbi. nih. gov/1000 genes/ftp /), Hapmap8 database (http:// Hapmap. ncbi. nlm. nih. gov /), and Yanhuang database (http:// yh. genomics. org. cn /), and the disease gene ERCC8 co-isolated from the disease haplotype was screened in this family using direct sequencing and the site was screened in the normal population peripheral blood genomic DNA samples without finding the mutation.

3. Direct sequencing method for searching ERCC8 gene mutation of patient in the family

PCR amplification of the fragment of interest: reaction conditions and reaction system:

(1) and (3) PCR reaction conditions: 5min at 95 ℃; 30sec at 94 ℃, 30sec (-1 ℃/cycles) at 60-50 ℃, 60sec at 72 ℃ and 10 cycles; 94 ℃ 30sec, 50 ℃ 30sec, 72 ℃ 60sec, 25 cycles; 10min at 72 ℃. The primer pair is ERCC 8-4F: GCTGACGTCATAGAGTCTTCA ERCC 8-4R: ACCACTGTACTGCTTTCACATC are provided.

(2) Reaction system: (TaKaRaTaq)

The reaction system is used for carrying out the amplification reaction of the genomic DNA template of each family member and the ERCC8 primer.

Sequencing a PCR product: the PCR products were sequenced by conventional Sanger sequencing method, and it was found that the bases 394-398 from the initiation codon in the ERCC8 gene coding region of two patients in the family were heterozygous deletion mutant, and the bases were changed from TTACA to- (FIG. 1). Multiple sequencing results indicated that the mutation site was not introduced due to amplification or sequencing errors. This mutation was not reported and was not present in the following four databases: the single nucleotide polymorphism database, the thousand-person genome project, the Hapmap8 database and the Yanhuang database indicate that the mutation is very rare, and the mutation causes the post-deletion frameshift of the 131 th amino acid of the ERCC8 protein. The mutation was found to be a "disease using" grade damage, causing the occurrence of congenital cataract and keratoconus in patients in this family, using the mutation prediction program MutationTaster (http:// www.mutationtaster.org/index. html). The site was screened for mutations in 200 samples of genomic DNA from peripheral blood of normal local population, and no mutations were found.

Designing a primer pair for amplifying each exon according to the genome sequence of ERCC8, wherein the sequence information of the positive primer and the negative primer is as follows:

ERCC8-1F:CGGTGTGAGGACACGATA

ERCC8-1R:AGGGCAGTTATTCTTTGGAG

ERCC8-2F:AAATACGTTAGGATGTGTGGTA

ERCC8-2R:CAACATTTGGTAGGTTCGATG

ERCC8-3F:AGAAATCTCTTTTGCCGTCT

ERCC8-3R:TATGTGAACCATTCGCTTGA

ERCC8-4F:GCTGACGTCATAGAGTCTTCA

ERCC8-4R:ACCACTGTACTGCTTTCACATC

ERCC8-5F:AAGAAGGGGCAATTCTAGGT

ERCC8-5R:TGAAGATGTTTGTTGCAGGT

ERCC6-6F:GCATGGATACAGTGAAAATGTC

ERCC8-6R:ACACTGATGTGAGTTGCATT

ERCC8-7F:TTTGGCCTCACTTTCTTCAG

ERCC8-7R:GACACAACACAGTTCCTCTG

ERCC8-8F:GGTACAGTCATTGTCCTAGC

ERCC8-8R:CTTTGTCACATGGATTGCAG

ERCC8-9F:CATGGCACAATTTCCTTGCT

ERCC8-9R:GTAGGTAGTGGGTAAGGGTG

ERCC8-10F:TAGCCAAATTACACTGCCAC

ERCC8-10R:ATATAACTGGTCTGGCAAGC

ERCC8-11F:GACATTGACATAGGCTGTGC

ERCC8-11R:TAGAAGTCACTGTACCATTTGTG

ERCC8-12F:AGTCTTAGCCTATGGATATCAG

ERCC8-12R:GTCACAACTGAGGTACAACG

each pair of primers described above was used to detect the ERCC8 gene.

In addition, one patient with congenital cataract and keratoconus emanation which is clearly diagnosed is collected in Shandong area, the peripheral blood genome DNA of the patient is extracted, the DNA template of the patient is applied to PCR amplification with ERCC8-4F and ERCC8-4R primers, the PCR product is sequenced by using a conventional Sanger sequencing method, the ERCC8 gene of the patient has SNP mutation discovered in the invention, namely, the base 394 to 398 from the start codon of the coding region has heterozygous deletion mutation, and the base is changed from TTACA to-.

Through the analysis, the ERCC8 gene can be used for detecting whether the patient is at risk of having congenital cataract and keratoconus. The risk of the disease of the person to be tested is determined by comparing each exon fragment of ERCC8 gene of the person to be tested with the normal corresponding fragment.

SEQUENCE LISTING

<110> institute of ophthalmology in Shandong province

Application of <120> ERCC8 gene in detection of congenital cataract combined with keratoconus

<130>

<160>24

<170>PatentIn version 3.5

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accactgtac tgctttcaca tc 22

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aagaaggggc aattctaggt 20

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atataactgg tctggcaagc 20

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Claims

1. The application of ERCC8 gene is characterized in that the application is the application of ERCC8 gene in preparing diagnostic products, and the diagnostic products are used for detecting SNP sites related to congenital cataract combined with keratoconus disease; the SNP locus is deletion mutation from 394 th to 398 th sites of an ERCC8 gene coding region from an initiation codon, and the base of the deletion mutation is TTACA;

the diagnostic product is a PCR detection primer;

the sequences of the PCR detection primers are SEQ ID NO. 7 and SEQ ID NO. 8.